The best choice of mass spectrometer for measuring the mass of large molecules, as undertaken particularly in biochemistry, is a time-of-flight mass spectrometer because it does not suffer from the limited mass range of other mass spectrometers. Time-of-flight mass spectrometers are frequently abbreviated to TOF or TOF-MS.
Two different types of time-of-flight mass spectrometer have been developed. The first type comprises time-of-flight mass spectrometers for measuring ions which are generated in pulses in a tiny volume and accelerated axially into the flight path, for example with ionization by matrix-assisted laser desorption, MALDI for short, a method of ionization suitable for ionizing large molecules.
The second type comprises time-of-flight mass spectrometers for the continuous injection of an ion beam, one section of which is ejected as a pulse in a “pulser” transversely to the direction of injection and forced to fly through the mass spectrometer as a linearly spread ion beam lying transverse to the direction of flight, as the schematic in FIG. 1 shows. A ribbon-shaped ion beam is therefore generated in which ions of the same type, i.e. with the same mass-to-charge ratio, form a transverse front. This second type of time-of-flight mass spectrometer is known for short as an “Orthogonal Time-of-Flight Mass Spectrometer” (OTOF); it is mainly used in conjunction with out-of-vacuum ionization. The most frequently used type of ionization is electrospray ionization (ESI). Electrospray ionization (ESI) is also suitable for ionizing large molecules. It is also possible to use other types of ionization, for example chemical ionization at atmospheric pressure (APCI), photoionization at atmospheric pressure (APPI) or matrix-assisted laser desorption at atmospheric pressure (AP-MALDI). Ions generated in-vacuum can also be used. Before they enter the OTOF, the ions can also be selected and fragmented in appropriate devices so that the fragments can be used to improve the characterization of the substances.
In this second type of time-of-flight mass spectrometer, a large number of spectra, each with relatively low ion counts, are generated by a very high number of pulses per unit of time (up to 20,000 pulses per second) in order to utilize the ions of the continuous ion beam as effectively as possible.
In devices that are nowadays commercially available, the scanning of the ion beams in these orthogonal time-of-flight mass spectrometers is carried out using what are referred to as channel plate secondary electron multipliers, whose individual pulses, triggered by the ions, are scanned by event counters with digitization of the event time (TDC: time to digital converter). This technique, however, only offers an extremely restricted dynamic measurement range, of the order of 1, and this can only be increased through summing a large number of individual spectra. The dynamic measurement range is defined as the ratio of the largest still undistorted signal recorded at the saturation limit to the smallest signal that can be distinguished from the background noise. Because of the restricted dynamic range of this TDC technology, newly developed equipment is now using fast transient recorders. The fast transient recorders digitize the amplified ion beams at a rate of between 1 and 4 gigahertz in analog-to-digital converters with a signal resolution of up to eight bits. This already gives the individual spectrum a dynamic measurement range of around 50; here again, however, a large number of individual spectra are added in order to reach higher dynamic measurement ranges.
Using a TDC, a spectrum with a dynamic range of 20,000 is achieved in one second, operating at 20,000 pulses per second. If, on the other hand, an ADC is used, the dynamic range rises to about 1,000,000. The use of ADCs, however, slightly reduces the mass resolution if good focusing achieves an ion beam signal width of about two nanoseconds.
As with all mass spectrometers, with a time-of-flight mass spectrometer one can only determine the ratio of the mass m of the ion to the number z of elementary charges which the ion carries. Any subsequent reference to “specific mass” or quite simply to “mass” on its own always means the ratio m/z. If, by way of exception, “mass” in the following text is to be taken to mean the physical dimension of the mass, it will be specifically called molecular mass. The unit of molecular mass m is the unified atomic mass unit, abbreviated to u, usually simply termed “mass unit” or “atomic mass unit”. In biochemistry and molecular biology, the essentially obsolete unit the dalton (“Da”) is frequently used. The unit of specific mass m/z is “mass unit per elementary charge” or “dalton per elementary charge”.
Electrospraying creates ions whose specific mass, m/z, hardly ever exceeds a value of around 5000 atomic mass units per elementary charge. This does not mean that only ions of molecules whose molecular weight does not exceed 5000 mass units can be ionized; molecules of larger mass are simply more frequently charged so that their specific mass, m/z, falls within this range. Ions of a molecule with 50 kilodaltons have a wide distribution of charge, z, extending from about 10 to 50 elementary charge units.
In a time-of-flight mass spectrometer having orthogonal ion injection, the ions in the pulser are accelerated transverse to the direction of their injection (the x-direction), and leave the pulser through openings in slit diaphragms. We refer to the direction of acceleration as the y-direction. After the acceleration, however, the ions are travelling in a direction in between the y-direction and the x-direction, because their original velocity in the x-direction is fully retained. The angle to the y-direction is given by a=arctan√Ex/Ey), where Ex is the kinetic energy of the ions in the primary beam in the x-direction, and Ey the energy of the ions following their acceleration in the y-direction. The direction in which the ions are flying after being pulsed out is independent of the mass of the ions.
The ions that have left the pulser then form a wide ribbon, in which ions of one type (one specific mass, m/z) each form a front that has the width of the beam in the pulser. Light ions fly faster, heavy ions fly more slowly, but all in the same direction, ignoring slight differences in direction that can arise as a result of slightly differing kinetic energies, Ex, of the ions as they are injected into the pulser. The field-free flight path must be entirely surrounded by the acceleration potential so that the flight of the ions is not disturbed.
Ions with the same specific mass which are at different locations of the beam cross section, and which therefore have different flight distances in front of them before reaching the detector, can be time-focused in reference to their different start locations. This is done by arranging that when the outpulsing voltage is switched on, the field in the pulser is selected so that the ions furthest away are given a somewhat higher acceleration energy, enabling them to catch up with the leading ions at a starting location focus point. The position of the starting location focus point can be freely selected through the outpulse field strength in the pulser.
In order to achieve a high resolution, the mass spectrometer is fitted with an energy-focusing reflector, which reflects the ion beam that has been pulsed out, across its whole width, toward the ion detector, thus giving ions of the same mass but with slightly different initial kinetic energies in the y-direction an accurate time-focus on the large-area detector. It is also possible for multiple reflectors to be used.
The ions fly away from the (last) reflector toward the detector, which must be as wide as the ion beam in order to be able to measure all the ions that arrive. This detector must also be aligned parallel to the x-direction, so that the front formed by flying ions of the same mass are detected at the same time.
Normally, a continuous beam of ions in the form of a fine ion beam is injected in the x-direction into the pulser. The ion velocity in the x-direction is then not changed, in spite of the perpendicular deflection. Following the lateral deflection in the y-direction and reflection in the reflector, the ions therefore reach the detector in the same time that they would have required to fly straight to the detector without the lateral deflection in the pulser (although they would not in fact then meet the detector, as they would be flying parallel to its surface).
Refilling the pulser after it has been emptied begins immediately after the ions have left the pulser. When the ions of the heaviest mass have flown far enough to have arrived at the detector, had the passage to the detector been free, then not only is the pulser full of the heaviest ions again, but the space between the pulser and the detector is also filled with ions. However, only those ions that are located in the pulser at the time of the next ejection pulse can be detected. The ions in the intermediate space between the pulser and the detector are lost for the purposes of analysis. It can be seen from this that, to achieve a high duty cycle for the ion beam, it is necessary to choose the geometry of the time-of-flight mass spectrometer in such a way that the detector is as close as possible to the pulser (with parallel reflection and detection: there are also other geometric arrangements).
The resolution, R, and the mass accuracy of a time-of-flight mass spectrometer are proportional to the flight distance. It is therefore possible to increase the resolution by providing a very long flight tube, or by introducing multiple reflections using several reflectors. It is possible, for instance, with a flight path of one and a half meters, to achieve a mass resolution of about R=m/Δm=10,000, and with around six meters a mass resolution of R=m/Δm=40,000 (where Δm is the line width of the ion signal at half maximum, measured in mass units). A long flight path, however, means that the pulse rate must be reduced to allow all the ions to reach the detector before the next pulse takes place. This, in turn, means that only a few ions in the ion beam are used for the measurement.
One known solution for achieving a high duty cycle of the ions together with the high resolution provided by a long flight path is intermediate storage of the ions in a storage ion guide, such as a guide hexapole. Intermediate storage in a quadruple, which can also be used for selection and fragmentation, is described in U.S. Pat. No. 6,285,027 B1 (I. Chernushevich and B. Thomson). Known methods can be used to store the ions here, and they are then driven out as required in order to fill the pulser. The disadvantage of this solution, however, is that the dynamic range of the measurements is greatly reduced. The fast transient recorders used nowadays, operating at one, two or four gigahertz, have an analog to digital converter with only eight bits of signal resolution, i.e. 256 counts, and the high data digitization rate restricts the signal resolution to between 5 and 7 bits. The dynamic range of the measurements of a single spectrum is therefore only roughly in the order of 50, in particular since the individual ions have to achieve at least a few counts in order to be detected above the noise. The saturation limit must not be exceeded in any of the individual spectra. A high dynamic range for the measurements can thus only be achieved by adding a large number of spectra. At least 2000 spectra must, for instance, be added together if a dynamic measurement range of 100,000 is desired, as is easily supplied by other types of mass spectrometry. If, however, the number of spectra per unit of time is reduced for high resolution, then the dynamic measurement range is also reduced to the same extent. If, instead of the analog to digital converters (ADC) mentioned here, only event time to digital converters (TDC) are used, as is usual in current commercial OTOFs, then another one or two orders of magnitude are lost from the dynamic measurement range, and this must be compensated for by adding a larger number of spectra together.
An insoluble dilemma is thus created: high mass resolution achieved by a long flight path means that an OTOF constructed according to conventional technology will always have either a low sensitivity or a low dynamic measurement range.